iTILLING: Personalized Mutation Screening

Abstract

One powerful approach to studying gene function is to analyze the phenotype of an organism carrying a mutant allele of a gene of interest. In order to use this experimental approach, one must have the ability to easily isolate individual organisms carrying desired mutations. A widely used method for accomplishing this task in plants and other organisms is a procedure called TILLING. A traditional TILLING project has at its foundation an ordered mutant population produced by treating seeds with a chemical mutagen. From this mutagenized seed, thousands of individual mutant lines are produced, and corresponding DNA samples are collected. For several plant species, publicly accessible screening facilities have been established that perform mutant screens on a gene-by-gene basis in response to customer requests using PCR and heteroduplex detection methods.

The iTILLING method described in this chapter represents an individualized version of the TILLING process. Performing a traditional TILLING experiment requires a large investment in time and resources to establish the well-ordered mutant population. By contrast, iTILLING is a low-investment alternative that provides the individual research lab with a practical solution to mutation screening. The main difference between the two approaches is that iTILLING is not based on the establishment of a durable, organized mutant population. Instead, a system for growing Arabidopsis seedlings in 96-well plates is used to produce an ephemeral mutant population for screening. Because the intention is not to develop a long-term resource, a considerable savings in time and money is realized when using iTILLING as compared to traditional TILLING. iTILLING is not intended to serve as a replacement to traditional TILLING. Rather, iTILLING provides a strategy by which custom mutagenesis screens can be performed by individual labs using unique genetic backgrounds that are of specific interest to that research group.